Discoveries during the prior period of support demonstrated that PKCe is a key enzyme that down-regulates GABAA receptor function through three mechanisms: (1) modulation of allosteric sensitivity via phosphorylation of receptor subunits;(2) down-regulation of cell surface abundance, possibly through phosphorylation of the N-ethylmaleimide-sensitive factor (NSF), and (3) down-regulation of GABAA receptor function. In this continuation, we will extend this work by examining PKCe phosphorylation of 32 and y2S GABAA receptor subunits, the regulation of NSF by PKCe, the role of cofilin-1 and cyclophilin A as possible PKCe substrates that regulate GABAA receptor sensitivity to allosteric drugs, and the role of increased a2 subunit abundance in the striatum in mediating PKCe null phenotypes. Studies will use transfected cell lines expressing specific receptor subunit combinations and an analog-sensitive mutant of PKCs. Other studies will use primary neurons cultured from PKCe null and wild type mice. Phosphorylation sites will be identified by mass spectrometry and phosphorylation site alanine mutants will be constructed to determine if these mutations prevent phosphorylation by PKCe. GABAA receptor currents in intact cells will be examined by whole cell patch clamp. a2 subunits will be overexpressed in the striatum to see if overexpression reduces alcohol consumption in wild type mice. An RNAi vector will be developed to knock down ot2in the striatum to determine if decreasing expressing of a2 subunits increases alcohol consumption in PKCe null mice. These studies will provide novel molecular information about how PKCe regulates the sensitivity of GABAA receptors to ethanol and will provide new information about how PKCe regulates GABAA receptor cell surface s1 ability and function. PERFORMANCE SITE(S) (organization, city, state) Ernest Gallo Clinic and Research Center Emeryville, CA PHS 398 (Rev. 09/04) Page 2 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Messing, Robert O. KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Messing, Robert O. ROBERM Ernest Gallo Clinic and Principal Investigator Research Center (EGCRQ Chou, Wen-Hai N/A EGCRC Assoc. Investigator Qi, Zhan-Heng N/A EGCRC Postdoctoral Fellow OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Shokat, Kevan M. UCSF Collaborator Human Embryonic Stem Cells ^ No (H Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list http://stemcells.nih.qov/reqistrv/index.asp. Usecontinuation pages as needed. If a specific line cannot be referencedat this time, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement. Applicable toSBIR/STTR Only. See SBIR/STTR instructions. Cl Yes d No PHS 398 (Rev. 09/04) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Messing, Robert O. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,